Humanization of an anti-carcinoembryonic antigen anti-idiotype antibody and use as a tumor vaccine and for targeting applications

ABSTRACT

A humanized form of an anti-idotype antibody to CEA, e.g., hW12, has conserved immunoreactivity. The clinical benefits of anti-CEA antibodies are maximized by using the humanized anti-idotype as a clearing agent for anti-CEA antibodies or antibody fragments. The humanized anti-idotype also can be used as an immunogenic vaccine.

The present application claims priority under 35 U.S.C. §371 toPCT/US97/04696 filed on Mar. 19, 1997, which claimed priority to U.S.Serial No. 60/013,708 filed on Mar. 20, 1996.

BACKGROUND OF THE INVENTION

The present invention relates to chimeric and humanized anti-idiotypeantibodies that specifically bind carcinoembryonic antigen antibodies.The invention further relates to the uses of the chimeric and humanizedanti-idiotype antibodies as clearing agents in therapeutic methods, as avaccine, and to detect in biological fluid samples the presence of anantibody or fragment thereof that specifically binds CEA.

The use of targeting monoclonal antibodies conjugated to radionuclidesor other cytotoxic agents offers the possibility of delivering suchagents directly to the tumor site, thereby limiting the exposure ofnormal tissues to toxic agents. (Goldenberg, Semin. Nucl. Med., 19:332(1989)). In recent years, the potential of antibody-based therapy andits accuracy in the localization of tumor-associated antigens have beendemonstrated both in the laboratory and clinical studies (see., e.g.,Thorpe, TIBTECH, 11:42 (1993); Goldenberg, Scientific American, Science& Medicine, 1:64 (1994); Baldwin et al., U.S. Pat. Nos. 4,925,922 and4,916,213; Young, U.S. Pat. No. 4,918,163; U.S. Pat. No. 5,204,095; Irieet al., U.S. Pat. No. 5,196,337; Hellstrom et al., U.S. Pat. Nos.5,134,075 and 5,171,665). In general, the use of radiolabeled antibodiesor antibody fragments against tumor-associated markers for localizationof tumors has been more successful than for therapy, in part becauseantibody uptake by the tumor is generally low, ranging from only 0.01%to 0.001% of the total dose injected (Vaughan et al., Brit. J. Radiol.,60:567 (1987)). Increasing the concentration of the radiolabel toincrease the dosage to the tumor is counterproductive generally as thisalso increases exposure of healthy tissue to radioactivity.

Carcinoembryonic antigen (CEA) is a 180,000-Da glycoprotein expressed inmost adenocarcinomas of endodermal-derived digestive-system epitheliaand in some other types of cancer such as breast cancer andnon-small-cell lung cancer. One of the main advantages of the CEA systemis that AB1 anti-CEA have been extensively used as radioimmunodetectionagents in cancer patients. One such antibody, MN-14, is a murine IgG₁Kmonoclonal antibody (Mab) with high affinity (K_(D)=10⁻⁹M) for humanCEA. In cancer patients, ¹³¹I-MN-14 targets CEA-producing tumorseffectively, and radiolabled MN-14-Fab′ can detect lesions as small as 2cm in diameter.

Rat anti-idiotype antibody (rWI2) against an anti-carcinoembryonicantigen antibody (MN-14) has been considered as a potential idiotypevaccine, capable of eliciting an Ab3 response in immunized animals.Losman et al., Int. J. Cancer 56:580-584 (1994). It has also been shownthat WI2 can serve as an effective clearing agent improvingtumor/nontumor ratios and reducing myelotoxicity, when used to removeexcess radiolabeled MN-14, as shown, for example, in U.S. Pat. No.4,624,846, the entire contents of which are incorporated herein byreference. However, its use is limited by the short biological half lifeof the rat Ab, due to rejection by the human host.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a chimericanti-idiotype Ab with anti carcinoembryonic-antibody properties, ahumanized anti-idiotype Ab with anti carcinoembryonic-antibodyproperties as an immunological reagent useful in clearing an organism ofanti-CEA antibody initially used as a cancer treatment, diagnostic, orvaccine, where the anti-idiotype Ab has the immunogenic properties of ahuman MAb in a human patient, and to provide an anti-idiotype Ab withanti carcinoembryonic-antibody properties which can serve as a detectionagent or vaccine. It is another object of the present invention toprovide DNA constructs encoding such antibodies.

To achieve these objectives, in one aspect of the invention, a chimericanti-idiotype antibody or fragment thereof which specifically binds tothe idiotype region of an anti-CEA monoclonal antibody is provided,comprising the rWI2 light chain and heavy chain variable regions, orsilent mutations thereof. In a preferred embodiment, the heavy chainvariable region comprises the ratWI2VK sequence (SEQ ID NO:18) shown inFIG. 1 and the light chain variable region comprises the RatWI2VKsequence (SEQ ID NO:22) shown in FIG. 2.

In another aspect of the invention, a humanized anti-idiotype antibodyor fragment thereof which specifically binds the idiotype region of ananti-CEA monoclonal antibody is provided, comprising rWI2 CDR regionsand humanized FR regions. In a preferred embodiment, the heavy chainvariable region comprises the KOLWI2VH-1 or the KOLWI2VH-2 sequence (SEQID NOS:19 or 20 respectively) shown in FIG. 1 and the light chainvariable region comprises the REIWI2VK or the REIWI2VKRS sequence (SEQID NOS:22 or 23 respectively) shown in FIG. 2.

In another aspect of the invention, an isolated polynucleotide encodingthe heavy chain or the heavy chain variable region of a chimeric orhumanized antibody or antibody fragment which specifically binds theidiotype region of an anti-CEA monoclonal antibody is provided,comprising sequences encoding at least two rWI2 heavy chain CDRs,selected from the group of CDRs consisting of:

the complementary determining region -1 (CDR-1) sequence (SEQ ID NO:1)NYWMT,

the complementary determining region -2 (CDR-2) sequenceSITSTGGTYHAESVKG, (SEQ ID NO:2) and

the complementary determining region -3 (CDR-3) sequence DDYGGQSTYVMDA(SEQ ID NO:3).

In another aspect of the invention, an isolated polynucleotide encodingthe light chain or the light chain variable region of a chimeric orhumanized antibody or antibody fragment which specifically binds theidiotype region of an anti-CEA monoclonal antibody is provided,comprising sequences encoding at least two rWI2 CDRs, selected from thegroup of CDRs consisting of:

the complementary determining region -1 (CDR1) sequence RASQDIGNYLR (SEQID NO:4),

the complementary determining region -2 (CDR2) sequence GATNLAA (SEQ IDNO:5), and

the complementary determining region -3 (CDR3) sequence LHHSEYPYT (SEQIS NO:6).

In another aspect of the invention, an isolated first expression vectorcomprising a gene for the chimeric WI2 heavy chain and an isolatedsecond expression vector comprising a gene for the chimeric WI2 lightchain are provided.

In another aspect of the invention, an isolated first expression vectorcomprising a gene for a humanized WI2 heavy chain and an isolated secondexpression vector comprising a gene for a humanized WI2 light chain areprovided.

In another aspect of the invention, a host is provided, comprising anisolated first vector comprising a gene for the chimeric WI2 heavy chainand an isolated second expression vector comprising a gene for thechimeric WI2 light chain, or an isolated first expression vectorcomprising a gene for a humanized WI2 heavy chain and an isolated secondexpression vector comprising a gene for a humanized WI2 light chain.

In another aspect of the invention, a method of stimulating an immuneresponse in a patient against cancers expressing carcinoembryonicantigen is provided, which comprises administering to a patient aneffective amount of a vaccine comprising a humanized anti-idiotypeantibody or fragment which specifically binds the idiotype region of ananti-CEA monoclonal antibody, conjugated to a soluble immunogeniccarrier protein, optionally in combination with a pharmaceuticallyacceptable vaccine adjuvant.

In another aspect of the invention, a method of diagnosis or treatmentof a patient is provided, wherein an antibody or antibody fragment thatspecifically binds CEA is used as a targeting, pre-targeting or therapyagent, either as such or as a component of a conjugate, the improvementto the method consisting of an anti-idiotype antibody used to clearnon-targeted antibody or antibody fragment.

In another aspect of the invention, a method of detecting the presenceof an antibody or fragment that specifically binds CEA, in a fluidbiological sample, comprising contacting the sample with rWI2, or achimeric anti-idiotype antibody or antibody fragment which specificallybinds the idiotype region of an anti-CEA monoclonal antibody, or ahumanized anti-idiotype antibody or antibody fragment which specificallybinds the idiotype region of an anti-CEA monoclonal antibody, anddetecting binding of said anti-idiotype antibody or antibody fragment toan antibody idiotype or antibody idiotype fragment in said sample.

DESCRIPTION OF THE FIGURES

FIG. 1 (SEQ ID NO:17-20, respectively) shows the design of the hVH. TherVH is shown aligned with KOL, and with the designed KOLW2VH-1 and 2sequences. Dashes indicate that the sequence matches at that positionthe rWI2VK sequence. Narrow boxes indicate positions where the rat aawas retained in the humanized FR sequences. Note that the KOLWI2VK-1sequence contains an extra rat aa, when compared with KOLWI2VK-2, atposition 5. CDRs are indicated by wider boxes and are labeled above therespective box as CDR1 to CDR3.

FIG. 2 (SEQ ID NO:21-24, respectively) shows the design of hVK. TheRATWI2VK sequence is shown aligned with REI, and with the designedREIWI2VK and REIWI2VKRS. Dashes indicate that the sequence matches atthat position the RATWI2VK sequence. Narrow boxes indicate positionswhere the rat aa was retained in the humanized FR sequences. Asindicated, four rat aa residues where retained in the FR regions. CDRsare indicated by wider boxes and are labeled above the respective box asCDR1-3.

FIGS. 3A-& B (SEQ ID NOS 25 & 26, respectively) shows the polynucleotidesequence for the hWI2 heavy chain variable region. The PCR primersemployed and the synthesized oligo K and oligo L described in the textare indicated. A single aa letter code is given below the polynucleotidesequence to represent the translation product. The CDRs are underlinedon the protein sequence.

FIGS. 4A & B (SEQ ID NOS 27 & 28, respectively) shows the polynucleotidesequence for the hWI2 light chain variable region. The PCR primersemployed and the synthesized oligo M′ and oligo N described in the textare indicated. A single aa letter code is given below the polynucleotidesequence for the translation product. The CDRs are underlined on theprotein sequence.

FIG. 5 shows a comparison between hWI2(RS), hWI2, cWI2, and rWI2 incompetitive binding assays. Binding of each of these antibodies is shownas a percentage of inhibition of the binding by a peroxidase conjugatedMN-14 antibody.

FIG. 6 (SEQ ID NO:29 & 30 respectively) shows the nucleic acid sequencefor the variable region of rWI2 light chain. The protein translationproduct is indicated below the nucleic acid sequence, using one letteraa code. The aa residues representing CDRs 1-3 are underlined andlabeled.

FIG. 7 (SEQ ID NO:31 & 32 respectively) shows the nucleic acid sequencefor the variable region of rWI2 heavy chain. The protein translationproduct is indicated below the nucleic acid sequence, using one letteraa code. The aa residues representing CDRs 1-3 are underlined andlabeled.

ILLUSTRATIVE GLOSSARY

The following terms or abbreviations are used in the presentapplication. The meanings set out in this glossary are for illustrativepurposes only. The full meaning of the terms will be apparent to thoseof skill in the art.

“CDR” is used as an abbreviation for Complementary Determining Region.These are the regions within the variable regions of an antibody thatare primarily, but not exclusively, responsible for antigen-antibodybinding.

“FR” is an abbreviation for Framework Region. Broadly speaking, theseare the portions of the variable regions of an antibody which lieadjacent to or flank the CDRs. In general, these regions have more of astructural function that affects the conformation of the variable regionand are less directly responsible for the specific binding of antigen toantibody, although, nonetheless, the framework regions can affect theinteraction.

An “antibody-binding region” is that part of an antibody that overall isresponsible for maintaining the structure of the antibody that interactswith the antigen. Generally, that is the combined light and heavyvariable domains of an antibody.

As used herein, “Chimeric” refers to an antibody in which the variableregion is derived from a rat antibody and the constant region is derivedfrom a human antibody.

“Humanized” refers to a chimeric antibody as defined above, but in whichthe FR variable regions are modified to make them more similar insequence to a human antibody.

A promoter is a DNA sequence that directs the transcription of astructural gene. Typically, a promoter is located in the 5′ region of agene, proximal to the transcriptional start site of a structural gene.If a promoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. In contrast, the rate oftranscription is not regulated by an inducing agent if the promoter is aconstitutive promoter.

An isolated DNA molecule is a fragment of DNA that is not integrated inthe genomic DNA of an organism. For example, a cloned T cell receptorgene is a DNA fragment that has been separated from the genomic DNA of amammalian cell. Another example of an isolated DNA molecule is achemically-synthesized DNA molecule that is not integrated in thegenomic DNA of an organism.

Complementary DNA (cDNA) is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand.

The term expression refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage, that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

As used herein, a silent mutation is a change introduced to the codingsequence of an antibody gene, which results in the expression of anantibody altered in its aa sequence, but, which is otherwisesubstantially similar to the antibody whose sequence was not altered, inrespect to immune activity.

A tumor associated antigen is a protein normally not expressed, orexpressed at very low levels, by a normal counterpart. Examples of tumorassociated antigens include α-fetoprotein and carcinoembryonic antigen(CEA).

In the present context, an anti-CEA MAb is a class III MAb, as describedby Primus et al., Cancer Research 43:686 (1983) and by Primus et al.,U.S. Pat. No. 4,818,709, which are incorporated by reference.

As used herein, an Ab1 is an antibody that binds with a tumor associatedantigen.

An anti-idiotype antibody (Ab2), as used herein, is an antibody thatbinds with an Ab1. Importantly, an Ab2 binds with the variable region ofAb1 and thus, an Ab2 mimics an epitope of a tumor associated antigen oran epitope of an infectious agent associated antigen.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-CEA Mab (Ab1) fragment binds with CEA,while an Ab2 fragment binds with the variable region of the Ab1 andmimics an epitope of CEA.

The term “antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“sFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

As used herein, the term antibody component or the term antibody mayinclude both an entire antibody and an antibody fragment.

Vaccine as used in the present context is an antibody or antibodyfragment which is engineered to more effectively stimulate an immuneresponse. Typically, this is achieved by conjugation of the antibodycomponent to a soluble immunogenic carrier protein.

DETAILED DESCRIPTION OF THE INVENTION

An Overview. Providing Chimeric and Humanized WI2 Anti-IdiotypeAntibodies.

Rat WI2 (rWI2) is an anti-idiotype monoclonal antibody which binds theCDR of MN-14. See Losman et al., (1994) supra. It was previouslyproposed that PCR primer pairs may be designed which might isolate thevariable regions of rat antibodies from a RNA substrate. See Kutemeieret al., Hybridoma, 11:23-32 (1992). Similar approaches have beenemployed to isolate the variable regions of mouse antibodies. SeeOrlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989). In thepresent invention, a combination of PCR primers as described byKutemeier et. al., and by Orlandi et al., supra, are required to isolatethe rWI2 variable region. These primers could be used to isolate otherrat variable regions.

cDNA for the variable regions of both the light and heavy chains of rWI2are isolated and the sequence from a number of clones is confirmed. Therat variable regions are cloned into plasmid vectors, such that the ratvariable K and H regions are attached to respective human IgG constantregions. The plasmids are transfected into SP2/O cells, clonescomprising both plasmids are selected, and chimeric WI2 (cWI2) antibodyis expressed.

To humanize the variable regions, FR rat regions are replaced by thehuman IgG counterparts. The rat CDR regions are preserved, in order tomaintain the specificity of the antibody. However, to enhance stabilityand maintain specificity and binding affinity of the antibody, it isbest to employ a human sequence which is most similar to the newlyidentified rat FR sequences. Rat residues that are close to the CDRs, orknown from prior experience to be important for interactions with theCDRs, were retained in both humanized sequences.

For both the heavy and light chain, long oligonucleotides correspondingto the variable region are engineered to reflect the humanized design.The oligonucleotides replace the rat variable region in plasmidconstructs designated to produce full length human W12 (hWI2). Theeffectiveness of cWI2 or hWI2 are compared with that of rWI2 in assayswhere they compete with CEA for binding to a peroxidase conjugatedMN-14.

In order to express the cWI2 and the hWI2s, the rat and the humanizedvariable regions are inserted into one or separate plasmid vectors insuch a manner as to allow coexpression of full length light and heavyantibody chains. The vectors can be transfected into SP2/O cells.Selection of transfected cells, and eventual amplification of theclones, are based on the expression of the dihydrofoate reductase (DHFR)gene which is located on the same plasmid vector. The functionality ofvarious constructs is determined by binding assays. For example, finalhWI2 constructs were compared with both rWI2 and cWI2 in their abilityto bind MN-14—see FIG. 5.

Production of Monoclonal Ab1 and Ab2 Antibodies

Rodent monoclonal antibodies to specific antigens may be obtained bymethods known to those skilled in the art. See, for example, Kohler andMilstein, Nature 256:495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) [hereinafter “Coligan”]. Briefly, monoclonal antibodies can beobtained by injecting rats with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures. Thesetechniques are applicable for production of a monoclonal antibody fromother mammals injected with an antigen.

A wide variety of monoclonal antibodies against tumor associatedantigens or infectious agents have been developed. See, for example,Goldenberg et al., international application publication No. WO 91/11465(1991), and Goldenberg, international application publication No. WO94/04702 (1994), each of which is incorporated herein by reference inits entirety.

Polyclonal Ab2 can be prepared by immunizing animals with Ab1 orfragments, using standard techniques. See, for example, Green et al.,“Production of Polyclonal Antisera,” in METHODS IN MOLECULAR BIOLOGY:IMMUNOCHEMICAL PROTOCOLS, Manson (ed.), pages 1-12 (Humana Press 1992).Also, see Coligan at pages 2.4.1-2.4.7. Alternatively, monoclonal Ab2can be prepared using Ab1 or fragments as immunogen with the techniquesdescribed above.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

The Design of a Humanized Antibody

One antibody of the present invention is a “humanized” monoclonalantibody. That is, rat complementary determining regions are transferredfrom heavy and light variable chains of the rat immunoglobulin into avariable region designed to contain a number of aa residues found withinthe FR region in human IgG. Similar conversion of mouse/human chimericantibodies to a humanized antibody has been described before. Generaltechniques for cloning murine immunoglobulin variable domains aredescribed, for example, by the publication of Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833 (1989), which is incorporated by referencein its entirety. Techniques for producing humanized MAbs are described,for example, by Jones et al., Nature 321:522 (1986), Riechmann et al.,Nature 332:323 (1988), Verhoeyen et al., Science 239:1534 (1988), Carteret al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992), Sandhu, Crit. Rev.Biotech. 12:437 (1992), and Singer et al., J. Immun. 150:2844 (1993),each of which is hereby incorporated by reference.

Construction of cWI2 and hWI2.

The engineering of chimeric and humanized WI2 employs standard molecularbiology and cellular biology approaches, for example PCR reactions,sequencing of DNA, synthesis of long oligonucleotides, cloning,site-directed mutagenesis, transfection of vectors into cells,expression of chimeric and humanized antibodies, their purification andso on. The techniques employed are standard techniques, well known inthe art and well established. A person skilled in the art would have nodifficulty carrying out those techniques. All necessary materials arereadily available. Reference manuals describing these techniques arewidely available. For example, see Sambrook et al., Molecular Cloning: ALaboratory Manual, second ed., Cold Spring Harbor Press, (1989); Co etal., J. Immunol., 148:1149 (1992); Ausubel et al., eds., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, and updates, John Wiley & Sons, NY,(1987); “Protocols—Current Methods and Applications” White, ed., Methodsin Mol. Biol. (15), Humana Press, Totawa, N.J. (1993); Uhlmann, Gene71:29 (1998); Wosnick et al., Gene 60:115 (1988); and Huse, in ANTIBODYENGINEERING: A PRACTICAL GUIDE, Boerrbaeck, ed., W. H. Freeman & Co.,103-120 (1992).

The PCR primers used in the initial cloning of the rat variable regionare designed over regions of the sequence that are expected to berelatively conserved between human antibodies. Furthermore, an aminoacid (aa) can generally be represented by more than one codon, althoughthe codons often differ only in the third position. Such codons aresometimes referred to as “degenerate.” To design the PCR primer oneattempts to choose a protein sequence over which degenerate codons arelimited in number. The approach is further limited by the need to designover a conserved protein sequence, as discussed above. Therefore, often,the “primer” as designed actually consists of a mix of numerousmolecules If that differ in sequence at specific positions. Any oneprimer molecule, which would provide a perfect match for the specificmRNA substrate to be amplified is present as a minuscule fraction of theoverall primer mix. To enhance likelihood of success, a few primerpairs, i.e. covering different regions of the mRNA are generally tried.

One skilled in the art recognizes that alternative techniques arereadily available. For example, initial changes to the cloned ratvariable region could be introduced by any of a number of site specificmutagenesis protocols. Furthermore, it should be realized thatadditional aa changes in the variable and especially the FR regions maybe possible, with the expectation that there will be some changes whichwould be silent in nature. Silent changes are those replacements,deletion or addition of one or a small number of aa that would notsignificantly affect the binding affinity or specificity of the antibodyor antibody fragments thereof. Most obvious among such silent changeswould be the replacement of one aa by an aa of a similar size andchemical properties. Such changes are well known in the art and aregenerally referred to as a “conservative” aa substitution. For example,a leucine when replaced by an isoleucine would generally not be expectedto affect the structure of a protein. All such conserved aa changes, aswell as silent changes which do not significantly affect the bindingaffinity or specificity of the anti-carcinoembryonic antibody, arewithin the scope of the invention.

Expression of the Engineered cWI2 or hWI2

Genes encoding the antibodies of the invention are introduced viaexpression vectors into a host cell, for expression. In a preferredembodiment, the genes for both the light and heavy genes are introducedin a single expression vector, which is introduced in a host cell. Theexpression vectors generally contain drug markers for selection of thetransformed cell. A drug marker can furthermore be used to amplify thecopy number of nearby genes, resulting in a clone over expressing theantibody. For example, a vector expressing the light and heavy chains ofcWI2 or hWI2 were introduced into SP2/O cells on vectors containing theDHFR gene. The original clones were amplified after selection by growthon methotrexate (MTX).

It should be understood that alternative ways to coexpress the light andheavy chain genes are feasible. A skilled artisan could consider otherselection regimens, introduction of both the light and heavy chain geneson one plasmid or cotransformation with separate vectors encoding heightand heavy genes, and transfection of other cell lines. Furthermore,expression of the antibody in yet other systems is possible. Forexample, expression could occur in yeast. Alternatively, baculovirusescan be engineered with the light and heavy genes and expressed incultured cells, or used to infect an insect.

The antibodies require purification from their expression system andmedia by methods that are generally similar to methods described abovefor purification of MAbs from hybridomas.

Uses for the Antibodies of the Invention

Humanized monoclonal antibodies in accordance with the invention aresuitable for use in therapeutic methods. For example, MN-14 has beenproposed as a therapeutic agent or as a vaccine to stimulate Ab2. Thedosage of MN-14 conjugated to a drug can be enhanced if removal orclearing of unbound MN-14 is made possible by its binding, and aggregateformation, with hWI2. Similarly, MN-14 can be used as a vaccine,optionally by alternative applications with an anti-idiotype antibodyvaccine. Delivery of the Ab2 would be counterproductive in the presenceof excess, free-floating MN-14 vaccine. Here, hWI2, for example, wouldclear the MN-14 vaccine, to allow efficient administration of the Ab2vaccine.

In addition, hWI2 itself can be a vaccine. Generally, the antibodies andfragments of the present invention can be used as vaccines byconjugating the antibodies or fragments to a soluble immunogenic carrierprotein. Suitable carrier proteins include keyhole lympet hemocyanin,which is the preferred carrier protein. The antibodies and fragments canbe conjugated to the carrier protein using standard methods. See, forexample, Hancock et al., “Synthesis of Peptides for Use as Immunogen, ”in METHODS IN MOLECULAR BIOLOGY: IMMUNOCHEMICAL PROTOCOLS, Manson (ed.),pages 23-32 (Humana Press 1992).

A preferred vaccine composition comprises an antibody conjugate orfragment conjugate, and an adjuvant. Examples of suitable adjuvantinclude aluminum hydroxide and lipid. Methods of formulating vaccinecompositions are well-known to those of ordinary skill in the art. See,for example, Rola, “Immunizing Agents and Diagnostic skin Antigens,” inREMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, Gennaro (ed.), pages1389-1404 (Mack Publishing company 1990).

Additional pharmaceutical methods may be employed to control theduration of action of a vaccine in a therapeutic application. Controlledrelease preparations can be prepared through the use of polymers tocomplex or adsorb the antibodies or fragments. For example,bio-compatible polymers include matrices of poly (ethylene-co-vinylacetate) and matrices of a polyanhydride copolymer of a stearic aciddimer and sebacic acid. Sherwood et al., Bio/Technology 10:1446 (1992).The rate of release of an antibody or antibody fragment from such amatrix depends upon the molecular weight of the antibody or fragment,the amount of antibody or fragment within the matrix, and the size ofdispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990).

The anti-idiotype Ab of the invention can be used for the detection ofan anti-carcinoembryonic antibody. For example, rWI2, cWI2 and hWI2 canbe used in vitro to test the blood sample of a patient for the presenceof anti-carcinoembryonic antibody. Detection of MN-14 levels, when MN-14is used as a therapeutic agent or as a vaccine would be important.Presence of MN-14 can be detected by rWI2, cWI2, or hWI2. Similarly,hWI2 can be used in a patient to determine the presence of naturalanti-CEA Abs. To be useful in the detection of an anti-CEA Ab, theanti-idiotype Abs can be conjugated to a label. Suitable labels include,e.g., a radiolabel, an enzyme, or a fluorescent label. Such labelingagents and methods of conjugation are well known to one skilled in theart.

The antibody preparations of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby antibodies or antibody fragments are combined in amixture with a pharmaceutically acceptable carrier. A composition issaid to be a “pharmaceutically acceptable carrier” if its administrationcan be tolerated by a recipient mammal. Sterile phosphate-bufferedsaline is one example of a pharmaceutically acceptable carrier. Othersuitable carriers are well-known to those in the art. See, for example,Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990).

The antibodies or fragments may be administered to a mammalintravenously or subcutaneously. Moreover, the administration may be bycontinuous infusion or by single or multiple boluses. Preferably, anantibody vaccine is administered subcutaneously, while an antibodypreparation that is not a vaccine is administered intravenously. Ingeneral, the dosage of administered antibodies or fragments for humanswill vary depending upon such factors as the patient's age, weight,height, sex, general medical condition and previous medical history.Typically, it is desirable to provide the recipient with a dosage ofantibodies or fragments which is in the range of from about 1 pg/kg to10 mg/kg (amount of agent/body weight of patient), although a lower orhigher dosage also may be administered as circumstances dictate.

For purposes of therapy, antibodies or fragments are administered to amammal in a therapeutically effective amount. An antibody preparation issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient mammal. In particular, anantibody preparation of the present invention is physiologicallysignificant if its presence invokes a humoral and/or cellular immuneresponse in the recipient mammal.

The present invention, in addition to the specifically describedtechniques, cell lines, and vectors, relies on descriptions of saidtechniques, cell lines, and vectors presented in U.S. Pat. Nos.5,443,953, and 4,624,846 and in U.S. patent applications Ser. Nos.08/318,157, and 08/289,576, all of which are incorporated herein byreference in their entireties.

EXAMPLE 1 Humanization of the W12 Clone

The sequences encoding the WI2 VH and VK domains were obtained byreverse transcriptase followed by PCR reactions (RT-PCR) using RNAprepared from WI2 hybridomas as the templates. Since the WI2 antibodysequences were of rat origin, the Orlandi VH1FOR/BACK and VK1FOR/BACKprimers (SEQ ID NO:7-16, respectively) sets might not be useful fortheir PCR-cloning. See Orlandi et al., 1989, supra. Based on thesequences suggested by Kutemeier et al., supra, primers were synthesizedthe following sets of primers for the PCR cloning of the VH and VKdomains of WI2:

(RCH1) (SEQ ID NO:7) 5′-GAC GTA TAC CTG TGG TTT TCT G-3′, (RVH-1BACK)(SEQ ID NO:8) 5′-AGG TSM ARC TGC AGS AGT CWG G-3′, and (RVH-1FOR) (SEQID NO:9) 5′-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC-3′, where S = G+ C; M = A + C; R = A + G; and W = A + T, and (RK1) (SEQ ID NO:10)5′-GGA TGA TGT CTT ATG AAC AA-3′, (RVK-1BACK) (SEQ ID NO:11) 5′-CCA GTTCCG AGC TCG TGC TCA CCC AGT CTC CA-3′, (RVK-2BACK) (SEQ ID NO:12) 5′-CCAGTT CCC AGC TCC AGA TGA CCC AGT CTC CA-3′, (RVK-3BACK) (SEQ ID NO:13)5′-CCA GAT GTG AGC TCG TGA TGA CCC AGA CTC CA-3′, (RVK-4BACK) (SEQ IDNO:14) 5′-CCA GAT GTG AGC TCG TCA TGA CCC AGT CTC CA-3′, (RVK-5BACK)(SEQ ID NO:15) 5′-CCA GTT CCG AGC TCG TGA TGA CAC AGT CTC CA-3′, and(RVK-1FOR) (SEQ ID NO:16) 5′-GTT AGA TCT CCA GCT TGG TCC C-3′.

The Cloning of VH Sequence

First-strand cDNA was prepared from total RNA isolated from WI2hybridoma using random hexamers as the annealing primers. RCH1 (whichanneals to the rat IgG1 CH1 domain) and RVH-1BACK (which anneals to rat5′ VH region) were tried as a primer pair to PCR-amplify and isolate theVH sequence from the first-strand cDNA template, using standardprotocols. However, no PCR product was obtained. Unexpectedly, the RCH1primer, in conjunction with the Orlandi primer (SEQ ID NO:8) VH1BACK(5′-AGG TSM ARC TGC AGS AGT CWG G-3′) produced a PCR product of theexpected size. The PCR-amplified VH sequence was digested with therestriction enzymes PstI/BstEII and subcloned into the correspondingsites of the heavy chain staging vector, VHpBS. See Leung et al.,Hybridoma 13: 469 (1994). Six individual clones were sequenced andconfirmed to be identical to each other. RVH-1FOR which anneals to rat3′ VH region and RVH-1BACK also produced a PCR product of the expectedsize, which was not further analyzed. FIG. 7 shows the sequence of theheavy chain variable region.

The Cloning of VR Sequence

First-strand cDNA was prepared from total RNA isolated from WI2hybridoma using random hexamers as the annealing primers. The RK1 (whichanneals to rat CK domain) in conjunction with RVK-3BACK (which anneal tothe 5′ VK region), was used to PCR-amplify and isolate the VK region ofrWI2, employing standard PCR protocols. The expected 320 base pairs (bp)product was obtained. The PCR product was further analyzed. Other primerpairs were not tested. FIG. 6 shows the sequence of the light chainvariable region.

Restriction analysis of the 320 bp VK PCR product revealed the presenceof an additional PvuII site which would interfere with subsequentcloning into the PvuII/BcII sites of the staging vector, VKpBR. Id.Because the PCR product lacked the appropriate ends for the stagingvector, it was directly subcloned into the TA Cloning Vector(Invitrogen), which allowed for direct insertions of PCR DNA. Sixindividual clones were sequenced and all six were shown to be identical.Analysis of the deduced protein sequence indicated a functional VXdomain. To subclone the sequence into a staging vector, a new primer wasdesigned. In this primer, a PvuII compatible end, rather than the wholerestriction recognition site, was introduced so that the PCR productwould have on its 5′ end a compatible site for ligation into thecorresponding PvuII site within the VKPBR staging vector. The PCRproduct was digested with BglII and subcloned into the PvuII/BcIIcloning sites of the VKpBR staging vector. Id. The ligation wastransformed in E. coli, and plated on selective plates. Miniprep DNA wasprepared and analyzed. DNA from positive clones were sequenced to makesure that the joining region, at the PvuII site, was as expected. ThePCR-amplified VH and VK sequence for WI2 were excised from theirrespective staging vector by HindIII/BamHI restriction digestion and theisolated fragments were subcloned into their respective heavy and lightchain expression vectors, pG1g and pKh. Id. Chimeric WI2 (cWI2) waspurified from clones cotransfected with the heavy and light chainexpression vectors and the immunoreactivity of cWI2 was compared to thatof rat WI2. Results indicated that both antibodies inhibited the bindingof MN-14 onto CEA to a similar extent, confirming the functionality ofthe chimeric antibody, and the authenticity of the VH and VK sequences.

Design of the Humanized W12 Sequence

VH and VK Design

By comparing the sequence homology of a number of human IgG to the rWI2frame work region, the human KOL framework was chosen for grafting theheavy chain CDRs. Two versions of the humanized heavy chain designatedas KOLWI2VH-1 and KOLWI2VH-2 were designed. See FIG. 1. However, onlythe KOLWI2VH-1 was synthesized and tested. KOLWI2VH-1 differs fromKOLWI2VH-2 only by one amino acid at position 5(Q instead of V). Ratresidues that are close to the CDRs, or known from prior experience tobe important for interactions with the CDRs, were retained in bothhumanized sequences. A total of 5 rat residues were retained in the FRregion of the humanized WI2 heavy chain sequence KOLWI2VH-1. TheKOLWI2VH-2 sequence would have retained four rat aa residues.

The REI framework was chosen for grafting the light chain CDRs. Four ratresidues were retained in the FR region of the designed light chain. Thesequence of the designed light chain, REIWI2VK is shown in FIG. 2.

Gene Synthesis for Humanized WI2 VH and VK Domain

Using computer analysis the nucleotide sequences encoding the humanizedVH and VK domain of WI2 were assembled as follows.

hWI2 VH

The long oligo K (135-mer)(SEQ ID NO:34) was PCR-amplified using theflanking primers oligo 21 (45-mer)(SEQ ID NO:33) and oligo 22(54-mer)(SEQ ID NO:36). The PCR product for oligo K encodes for theN-terminal half of the VH domain. Similarly, the long oligo L(133-mer)(SEQ ID NO:37) was PCR-amplified using the flanking primersoligo 23 (48-mer)(SEQ ID NO:35) and oligo 24 (45-mer)(SEQ ID NO:38). ThePCR product for oligo L encodes for the C-terminal half of the VHdomain. The sequences of the different oligos used for the PCR-synthesisof the humanized WI2 VH domain are summarized in FIG. 3.

The PCR product for oligo K was digested with PstI/AlwNI, whereas thePCR product for oligo L was digested with AlwNI/BstEII. The K and Lfragments were gel-purified and ligated to the PstI/BstEII site of thestaging, vector VHpBR. Minipreps were analyzed by restriction analysisas well as by sequencing reaction. MP33#4(1.6.95) was confirmed to havethe correct sequence for the hWI2 VH, and was used for subsequentcloning and subcloning. The BaraHI/HindIII fragment containing the hWI2VH domain was gel-purified from the DNA after restriction digestion, andwas cloned into the corresponding sites of the heavy chain expressionvector, pG1g. Leung et al. Hybridoma 13:469 (1994).

hWI2 VK

The long oligo M′ (129-mer) (SEQ ID NO:41) was PCR-amplified using theflanking primers oligo 25 containing a PvuII site (SEQ ID NO:39 & 40,respectively) and oligo 26 (SEQ ID NO:43). The PCR product for oligo M′encodes for the N-terminal half of the VK domain. The long oligo N(128-mer)(SEQ ID NO:44) was PCR-amplified by the flanking primers oligo27 (44-mer)(SEQ ID NO:43) and oligo 28 (39-mer)(SEQ ID NO:45). The PCRproduct for oligo N encodes for the C-terminal half of the VK domain.The sequences of the oligonucleotides used for the PCR-amplification ofthe humanized WI2 VK domain are summarized in FIG. 4.

The PCR product for oligo M′ was digested with PstI and PvuII, whereasthe PCR product for oligo N was digested with PstI/BgIII. The M and Nfragments were gel-purified and ligated to the PvuII/BcII site of thestaging vector VKpBR. By analysis of miniprep DNA three clones wereidentified to be the likely positives for hWI2VKpBR. Hogness stocks forthese three minipreps were prepared. The sequence was confirmed fromboth directions, crossing the PvuII and BglII/BcII junctions. PlasmidDNA was prepared. The BamHI/HindIII fragment containing the hWI2 VKdomain was gel-purified from the DNA after restriction digestion, andwas cloned into the corresponding sites of the light chain expressionvector, pKh. Id. Plasmid DNA for both the heavy and light chain vectorsexpressing hWI2 were prepared and cotransfected into SP2/O myeloma cellsby electroporation.

A total of eight antibody producing colonies were identified using ELISAassays. The three highest producers were maintained and cell aliquotswere frozen for storage. The highest producer was expanded and upscaledfor subsequent antibody purification and testing.

Examining the Immunoreactivity of hWI2 Heavy Chain in a Mix-and-MatchExperiment

The mix-and-match antibody containing the humanized heavy and chimericlight chains of WI2 was purified. Various concentrations of themix-and-match antibody were used to compete with ELISA-plate coated CEAfor binding to the peroxidase-conjugated MN-14 antibody. Chimeric Ab wasproduced and expressed and shown to have immunogenic reactivitycomparable to that of the rat WI2. The immunoreactivity of themix-and-match WI2 was compared to that of cWI2, which has been confirmedto be identical to that of rat WI2. The humanized heavy chain for WI2maintained the original immunoreactivity of rat WI2.

WI2

The original sequences for humanized WI2 heavy and light chains weredesigned from sequence homology comparison without the aid of computermodeling (FIG. 2). In a competitive binding assay, the first version ofhumanized WI2 (hWI2) was shown to be slightly less effective in blockingthe binding of peroxidase conjugated MN-14 to CEA, as compared to thatof rWI2 and cWI2 (FIG. 5). In the mix-and-match experiment (rVK+hVH)described above, it was demonstrated that the design of the humanizedheavy chain was good. Therefore, a redesign of the humanized VK sequencewas undertaken in an attempt to restore the full immunoreactivity ofhWI2. Studies on the crystal structure for REI protein suggested thatthe rat residues Arg (66) and Ser (69) might be interacting with thelight chain CDRs. These two rat residues, arg and ser were reintroducedinto the VK FR3 of hWI2 by site-directed mutagenesis. The newly designedlight chain, REIWI2VKRS is shown in FIG. 2. Humanized WI2 containingthese two mutations was designated as hWI2RS. All mutations and vectorconstructions were confirmed by sequencing as well as by extensiverestriction analysis. Expression vectors for this new version ofhumanized light chain (hWl2RSpKh) were used for transfection experimentsby electroporation.

For unknown reasons, it had been difficult to get antibody producingclones transfected with the hWI2RS expression vector. Clones 131A9 and132F7 were identified as positive clones producing about 0.4 and 0.2mg/liter of hWI2RS antibodies, respectively. One liter cultures of clone131A9 and 132F7 were grown in roller bottles and the antibody waspurified by a standard protein purification method. Approximately 0.4and 0.2 mg of hWI2RS was obtained from clone 131A9 and 132F7,respectively. The immunoreactivities of 131A9 and 132F7 hWI2RS, asmeasured by their abilities to block MN-14 binding on CEA, were shown tobe similar.

EXAMPLE 2 Comparing the Immuuoreactivities of Different Versions of WI2

In one experiment, the immunoreactivities of hWI2 and hWI2RS, asmeasured by their abilities to block MN-14 binding on CEA, were comparedto that of rWI2 and cWI2, respectively (FIG. 5). Both hWI2 and hWI2RSappeared to exhibit a slightly reduced immunoreactivities than that ofrWI2 and cWI2. The introduction of the RS mutation into the FR3 regionof hWI2 did not seem to improve immunoreactivity as predicted.Nevertheless, hWI2 was nearly as immunoreactive as rWI2 or cWI2.

Both hWI2 and hWI2RS exhibited very similar, if not identical,immunoreactivity. Since hWI2RS contains two extra rat residues in thelight chain FR3 region, and would be potentially more immunogenic thefirst humanized version of WI2, hWI2, was chosen to be the antibody thatwas further scaled up for production.

EXAMPLE 3 Construction of an Amplifiable Expression Vector for hWI2

The VH and VK sequence for hWI2 were excised from the respective stagingvectors and ligated to a single amplifiable vector, pdHL2. The pdHL2vector contains sequences for the human CK, IgG1, and an amplifiableDHFR gene, each controlled by separate promoters. See Leung et al.,Tumor Targeting 2:184 (195) (1996) and Losman et al., Tumor Targeting2:183 (#93) (1996). The resultant vector expressing the hWI2 wasdesignated hWI2pdHL2. The plasmid DNA for hWI2pdHL2 was linearized andtransfected by electroporation into SP2/O cells. Selection was performedby the addition of 0.1 μM of methotrexate (MTX) into the culture media.Amplification was carried out in a stepwise fashion with increasingconcentration of MTX (from 0.1 to 3 μM). hWI2 purified from amplifiedclones exhibited identical immunoreactivity as hWI2 obtained fromprevious non-amplifiable clones.

45 5 amino acids amino acid linear peptide 1 Asn Tyr Trp Met Thr 1 5 16amino acids amino acid linear peptide 2 Ser Ile Thr Ser Thr Gly Gly ThrTyr His Ala Glu Ser Val Lys Gly 1 5 10 15 13 amino acids amino acidlinear peptide 3 Asp Asp Tyr Gly Gly Gln Ser Thr Tyr Val Met Asp Ala 1 510 11 amino acids amino acid linear peptide 4 Arg Ala Ser Gln Asp IleGly Asn Tyr Leu Arg 1 5 10 7 amino acids amino acid linear peptide 5 GlyAla Thr Asn Leu Ala Ala 1 5 9 amino acids amino acid linear peptide 6Leu His His Ser Glu Tyr Pro Tyr Thr 1 5 22 base pairs nucleic acidsingle linear other nucleic acid /desc = “primer” 7 GACGTATACCTGTGGTTTTC TG 22 22 base pairs nucleic acid single linear other nucleicacid /desc = “primer” 8 AGGTSMARCT GCAGSAGTCW GG 22 32 base pairsnucleic acid single linear other nucleic acid /desc = “primer” 9TGAGGAGACG GTGACCGTGG TCCCTTGGCC CC 32 20 base pairs nucleic acid singlelinear other nucleic acid /desc = “primer” 10 GGATGATGTC TTATGAACAA 2032 base pairs nucleic acid single linear other nucleic acid /desc =“primer” 11 CCAGTTCCGA GCTCGTGCTC ACCCAGTCTC CA 32 32 base pairs nucleicacid single linear other nucleic acid /desc = “primer” 12 CCAGTTCCGAGCTCCAGATG ACCCAGTCTC CA 32 32 base pairs nucleic acid single linearother nucleic acid /desc = “primer” 13 CCAGATGTGA GCTCGTGATG ACCCAGACTCCA 32 32 base pairs nucleic acid single linear other nucleic acid /desc= “primer” 14 CCAGATGTGA GCTCGTCATG ACCCAGTCTC CA 32 32 base pairsnucleic acid single linear other nucleic acid /desc = “primer” 15CCAGTTCCGA GCTCGTGATG ACACAGTCTC CA 32 22 base pairs nucleic acid singlelinear other nucleic acid /desc = “primer” 16 GTTAGATCTC CAGCTTGGTC CC22 122 amino acids amino acid linear protein 17 Gln Val Gln Leu Val GluSer Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu SerCys Ser Ser Ser Gly Phe Ile Phe Ser Xaa Xaa 20 25 30 Xaa Xaa Xaa Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Arg Phe ThrIle Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met AspSer Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Arg Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp 100 105 110 Gly Gln GlyThr Pro Val Thr Val Ser Ser 115 120 122 amino acids amino acid linearprotein 18 Gln Val Gln Leu Gln Glu Ser Gly Gly Asp Leu Val Gln Pro GlyArg 1 5 10 15 Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe SerAsn Tyr 20 25 30 Trp Met Thr Trp Ile Arg Gln Ala Pro Gly Glu Gly Leu GluTrp Val 35 40 45 Ala Ser Ile Thr Ser Thr Gly Gly Gly Thr Tyr His Ala GluSer Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser ThrLeu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala ThrTyr Tyr Cys 85 90 95 Ser Arg Asp Asp Tyr Gly Gly Gln Ser Thr Tyr Val MetAsp Ala Trp 100 105 110 Gly Gln Gly Ser Ser Val Thr Val Ser Ser 115 120122 amino acids amino acid linear protein 19 Gln Val Gln Leu Gln Glu SerGly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser CysSer Ser Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp Ile ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Thr Ser ThrGly Gly Gly Thr Tyr His Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr IleSer Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asp SerLeu Arg Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ser Arg Asp Asp TyrGly Gly Gln Ser Thr Tyr Val Met Asp Ala Trp 100 105 110 Gly Gln Gly ThrPro Val Thr Val Ser Ser 115 120 122 amino acids amino acid linearprotein 20 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ser Ser Gly Phe Thr Phe SerAsn Tyr 20 25 30 Trp Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val 35 40 45 Ala Ser Ile Thr Ser Thr Gly Gly Gly Thr Tyr His Ala GluSer Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn ThrLeu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly ValTyr Tyr Cys 85 90 95 Ser Arg Asp Asp Tyr Gly Gly Gln Ser Thr Tyr Val MetAsp Ala Trp 100 105 110 Gly Gln Gly Thr Pro Val Thr Val Ser Ser 115 120109 amino acids amino acid linear protein 21 Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile ThrCys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Trp Tyr Gln GlnThr Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Xaa Xaa Xaa Xaa XaaXaa Xaa Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr AspTyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala ThrTyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Phe Gly Gln GlyThr Lys Leu Gln Ile Thr Arg Thr 100 105 108 amino acids amino acidlinear protein 22 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Pro AlaSer Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln AspIle Gly Asn Tyr 20 25 30 Leu Arg Trp Phe Gln Gln Lys Pro Gly Lys Ser ProArg Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Ala Gly Val Pro SerArg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Phe Ser Leu Thr Ile AsnSer Leu Glu Ser 65 70 75 80 Glu Asp Met Ala Ile Tyr Tyr Cys Leu His HisSer Glu Tyr Pro Tyr 85 90 95 Thr Phe Gly Ile Gly Thr Lys Leu Glu Arg LysArg 100 105 108 amino acids amino acid linear protein 23 Asp Ile Gln MetThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Asn Tyr 20 25 30 Leu Arg TrpPhe Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly AlaThr Asn Leu Ala Ala Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg SerGly Ser Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu AspIle Ala Thr Tyr Tyr Cys Leu His His Ser Glu Tyr Pro Tyr 85 90 95 Thr PheGly Ile Gly Thr Lys Leu Gln Ile Lys Arg 100 105 108 amino acids aminoacid linear protein 24 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser GlnAsp Ile Gly Asn Tyr 20 25 30 Leu Arg Trp Phe Gln Gln Thr Pro Gly Lys AlaPro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Ala Gly Val ProSer Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr IleSer Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu HisHis Ser Glu Tyr Pro Tyr 85 90 95 Thr Phe Gly Ile Gly Thr Lys Leu Gln IleLys Arg 100 105 366 base pairs nucleic acid double linear cDNA CDS1..366 25 CAG GTC CAA CTG CAG GAG TCA GGG GGA GGT GTA GTG CAG CCT GGAAGG 48 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 15 10 15 TCT CTG AGA CTT TCC TGT AGC TCA TCT GGA TTC ACA TTC AGT AAT TAC96 Ser Leu Arg Leu Ser Cys Ser Ser Ser Gly Phe Thr Phe Ser Asn Tyr 20 2530 TGG ATG ACT TGG ATA CGC CAG GCT CCA GGG AAG GGT CTT GAA TGG GTT 144Trp Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45GCG TCC ATT ACT AGT ACT GGT GGT GGT ACC TAC CAT GCA GAG TCT GTG 192 AlaSer Ile Thr Ser Thr Gly Gly Gly Thr Tyr His Ala Glu Ser Val 50 55 60 AAGGGC CGA TTC ACT ATC TCC AGA GAT AAT TCA AAA AAC ACC CTG TTC 240 Lys GlyArg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 CTGCAA ATG GAC AGT CTG AGG CCT GAG GAC ACG GGC GTT TAT TAC TGT 288 Leu GlnMet Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 TCA AGAGAT GAC TAC GGA GGA CAG AGC ACC TAT GTT ATG GAT GCC TGG 336 Ser Arg AspAsp Tyr Gly Gly Gln Ser Thr Tyr Val Met Asp Ala Trp 100 105 110 GGT CAGGGA ACT CCG GTC ACC GTC TCC TCC 366 Gly Gln Gly Thr Pro Val Thr Val SerSer 115 120 122 amino acids amino acid linear protein 26 Gln Val Gln LeuGln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu ArgLeu Ser Cys Ser Ser Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met ThrTrp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser IleThr Ser Thr Gly Gly Gly Thr Tyr His Ala Glu Ser Val 50 55 60 Lys Gly ArgPhe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu GlnMet Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ser ArgAsp Asp Tyr Gly Gly Gln Ser Thr Tyr Val Met Asp Ala Trp 100 105 110 GlyGln Gly Thr Pro Val Thr Val Ser Ser 115 120 325 base pairs nucleic aciddouble linear cDNA CDS 1..324 27 GAC ATT CAG ATG ACC CAG TCT CCA TCT TCCCTG TCT GCG TCT GTG GGA 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser LeuSer Ala Ser Val Gly 1 5 10 15 GAC AGA GTC ACT ATT ACT TGC CGG GCA AGTCAA GAC ATT GGA AAT TAT 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser GlnAsp Ile Gly Asn Tyr 20 25 30 TTA AGA TGG TTC CAG CAG ACA CCG GGG AAA GCTCCG AAA CTT TTG ATT 144 Leu Arg Trp Phe Gln Gln Thr Pro Gly Lys Ala ProLys Leu Leu Ile 35 40 45 TAT GGT GCA ACC AAC TTG GCT GCA GGG GTC CCA TCACGG TTC AGT GGC 192 Tyr Gly Ala Thr Asn Leu Ala Ala Gly Val Pro Ser ArgPhe Ser Gly 50 55 60 AGT GGG TCT GGG ACA GAT TTT ACT TTT ACC ATC TCA AGCCTT CAG CCT 240 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser LeuGln Pro 65 70 75 80 GAA GAT ATT GCT ACT TAT TAC TGT CTG CAC CAT TCT GAGTAT CCA TAC 288 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu His His Ser Glu TyrPro Tyr 85 90 95 ACG TTT GGA ATT GGG ACC AAG TTG CAG ATC AAA CGT G 325Thr Phe Gly Ile Gly Thr Lys Leu Gln Ile Lys Arg 100 105 108 amino acidsamino acid linear protein 28 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser LeuSer Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala SerGln Asp Ile Gly Asn Tyr 20 25 30 Leu Arg Trp Phe Gln Gln Thr Pro Gly LysAla Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Ala Gly ValPro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe ThrIle Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys LeuHis His Ser Glu Tyr Pro Tyr 85 90 95 Thr Phe Gly Ile Gly Thr Lys Leu GlnIle Lys Arg 100 105 324 base pairs nucleic acid double linear cDNA CDS1..324 29 GAC ATT CAG CTG ACC CAG TCT CCA GCT TCC CTG CCT GCG TCT CTGGGA 48 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Pro Ala Ser Leu Gly 15 10 15 GAC AGA GTC ACT ATT ACT TGC CGG GCA AGT CAA GAC ATT GGA AAT TAT96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Asn Tyr 20 2530 TTA AGA TGG TTC CAG CAG AAA CCG GGG AAA TCT CCG AGG CTT TTG ATT 144Leu Arg Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Leu Leu Ile 35 40 45TAT GGT GCA ACC AAC TTG GCA GCT GGG GTC CCA TCA CGG TTC AGT CGC 192 TyrGly Ala Thr Asn Leu Ala Ala Gly Val Pro Ser Arg Phe Ser Arg 50 55 60 AGTAGG TCT GGG TCA GAT TTT TCT CTG ACC ATC AAC AGC CTG GAG TCT 240 Ser ArgSer Gly Ser Asp Phe Ser Leu Thr Ile Asn Ser Leu Glu Ser 65 70 75 80 GAAGAT ATG GCT ATT TAT TAC TGT CTG CAC CAT TCT GAG TAT CCA TAC 288 Glu AspMet Ala Ile Tyr Tyr Cys Leu His His Ser Glu Tyr Pro Tyr 85 90 95 ACG TTTGGA ATT GGG ACC AAG CTG GAA CGG AAA CGG 324 Thr Phe Gly Ile Gly Thr LysLeu Glu Arg Lys Arg 100 105 108 amino acids amino acid linear protein 30Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Pro Ala Ser Leu Gly 1 5 1015 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Asn Tyr 20 2530 Leu Arg Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Leu Leu Ile 35 4045 Tyr Gly Ala Thr Asn Leu Ala Ala Gly Val Pro Ser Arg Phe Ser Arg 50 5560 Ser Arg Ser Gly Ser Asp Phe Ser Leu Thr Ile Asn Ser Leu Glu Ser 65 7075 80 Glu Asp Met Ala Ile Tyr Tyr Cys Leu His His Ser Glu Tyr Pro Tyr 8590 95 Thr Phe Gly Ile Gly Thr Lys Leu Glu Arg Lys Arg 100 105 366 basepairs nucleic acid double linear cDNA CDS 1..366 31 CAG GTC CAA CTG CAGGAG TCA GGG GGA GAT CTA GTG CAG CCT GGA AGG 48 Gln Val Gln Leu Gln GluSer Gly Gly Asp Leu Val Gln Pro Gly Arg 1 5 10 15 TCT CTG AAA CTT TCCTGT GTA GCC TCT GGA TTC ACA TTC AGT AAT TAC 96 Ser Leu Lys Leu Ser CysVal Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 TGG ATG ACT TGG ATC CGCCAG GCT CCA GGG GAG GGT CTT GAA TGG GTT 144 Trp Met Thr Trp Ile Arg GlnAla Pro Gly Glu Gly Leu Glu Trp Val 35 40 45 GCG TCC ATT ACT AGT ACT GGTGGT GGG ACT TAC CAT GCA GAG TCT GTG 192 Ala Ser Ile Thr Ser Thr Gly GlyGly Thr Tyr His Ala Glu Ser Val 50 55 60 AAG GGC CGA TTC ACT ATC TCC AGAGAT AAT TCA AAA AGC ACC CTG TAC 240 Lys Gly Arg Phe Thr Ile Ser Arg AspAsn Ser Lys Ser Thr Leu Tyr 65 70 75 80 CTG CAA ATG AAC AGT CTG AGG CCTGAG GAC ACG GCC ACT TAT TAC TGT 288 Leu Gln Met Asn Ser Leu Arg Pro GluAsp Thr Ala Thr Tyr Tyr Cys 85 90 95 TCA AGA GAT GAC TAC GGA GGA CAG AGCACC TAT GTT ATG GAT GCC TGG 336 Ser Arg Asp Asp Tyr Gly Gly Gln Ser ThrTyr Val Met Asp Ala Trp 100 105 110 GGT CAG GGA TCT TCG GTC ACC GTC TCCTCA 366 Gly Gln Gly Ser Ser Val Thr Val Ser Ser 115 120 122 amino acidsamino acid linear protein 32 Gln Val Gln Leu Gln Glu Ser Gly Gly Asp LeuVal Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Val Ala Ser GlyPhe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Thr Trp Ile Arg Gln Ala Pro GlyGlu Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Thr Ser Thr Gly Gly Gly ThrTyr His Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp AsnSer Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro GluAsp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ser Arg Asp Asp Tyr Gly Gly Gln SerThr Tyr Val Met Asp Ala Trp 100 105 110 Gly Gln Gly Ser Ser Val Thr ValSer Ser 115 120 46 base pairs nucleic acid single linear other nucleicacid /desc = “oligo” 33 CAGGTCCAAC TGCAGGAGTC AGGGGGAGGT GTAGTGCAGCCTGGAA 46 130 base pairs nucleic acid single linear other nucleic acid/desc = “oligo” 34 AGTAATGGAC GCAACCCATT CAAGACCCTT CCCTGGAGCCTGGCGTATCC AAGTCATCCA 60 GTAATTACTG AATGTGAATC CAGATGAGCT ACAGGAAAGTCTCAGAGACC TTCCAGGCTG 120 CACTACACCT 130 48 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligo” 35 ATGCAGAGTCTGTGAAGGGC CGATTCACTA TCTCCAGAGA TAATTCAA 48 54 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligo” 36 TTCACAGACTCTGCATGGTA GGTACCACCA CCAGTACTAG TAATGGACGC AACC 54 133 base pairsnucleic acid single linear other nucleic acid /desc = “oligo” 37ACCCCAGGCA TCCATAACAT AGGTGCTCTG TCCTCCGTAG TCATCTCTTG AACAGTAATA 60AACGCCCGTG TCCTCAGGCC TCAGACTGTC CATTTGCAGG AACAGGGTGT TTTTTGAATT 120ATCTCTGGAG ATA 133 45 base pairs nucleic acid single linear othernucleic acid /desc = “oligo” 38 GGAGGAGACG GTGACCGGAG TTCCCTGACCCCAGGCATCC ATAAC 45 50 base pairs nucleic acid single linear othernucleic acid /desc = “oligo” 39 GACATTCAGC TGACCCAGTC TCCATCTTCCCTGTCTGCGT CTGTGGGAGA 50 41 base pairs nucleic acid single linear othernucleic acid /desc = “oligo” 40 ATGACCCAGT CTCCATCTTC CCTGTCTGCGTCTGTGGGAG A 41 129 base pairs nucleic acid single linear other nucleicacid /desc = “oligo” 41 AGTTGGTTGC ACCATAAATC AAAAGTTTCG GAGCTTTCCCCGGTGTCTGC TGGAACCATC 60 TTAAATAATT TCCAATGTCT TGACTTGCCC GGCAAGTAATAGTGACTCTG TCTCCCACAG 120 ACGCAGACA 129 44 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligo” 42 TGGCTGCAGGGGTCCCATCA CGGTTCAGTG GCAGTGGGTC TGGG 44 36 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligo” 43 GGACCCCTGCAGCCAAGTTG GTTGCACCAT AAATCA 36 128 base pairs nucleic acid singlelinear other nucleic acid /desc = “oligo” 44 TTGGTCCCAA TTCCAAACGTGTATGGATAC TCAGAATGGT GCAGACAGTA ATAAGTAGCA 60 ATATCTTCAG GCTGAAGGCTTGAGATGGTA AAAGTAAAAT CTGTCCCAGA CCCACTGCCA 120 CTGAACCG 128 39 basepairs nucleic acid single linear other nucleic acid /desc = “oligo” 45CACGTTAGAT CTGCAACTTG GTCCCAATTC CAAACGTGT 39

What is claimed is:
 1. An anti-idiotype antibody or an antigen bindingfragment thereof, which specifically binds to the idiotype region of ananti-CEA monoclonal antibody, MN-14, wherein said anti-idiotypicantibody comprises the rat anti-idiotypic antibody (rWI2) light chainvariable region comprising SEQ ID NO:22 and heavy chain variable regioncomprising SEQ ID NO: 18 derived from a rat antibody and constantregions selected from human antibody sequences.
 2. An antibody orfragment thereof according to claim 1, wherein said anti-idiotypeantibody or antibody fragment is labeled with a radiolabel, an enzyme,or a fluorescent agent.
 3. A humanized anti-idiotype antibody or anantigen binding fragment thereof which specifically binds the idiotyperegion of an anti-CEA CEA monoclonal antibody, MN-14, wherein saidhumanized anti-idiotypic antibody comprises rat anti-idiotypic antibody(rWI2) CDRs, wherein the CDRs of the light chain variable regioncomprises: the CDR-1 sequence, SEQ ID NO: 4, RASQDIGNYLR, the CDR-2sequence, SEQ ID NO: 5, GATNLAA, and the CDR-3 sequence, SEQ ID NO: 6,LHHSEYPYT; and, wherein the CDRs of the heavy chain variable regioncomprises: the CDR-1 sequence, SEQ ID NO: 1, NYWMT, the CDR-2 sequence,SEQ ID NO: 2, SITSTGGTYHAESVKG, and the CDR-3 sequence, SEQ ID NO: 3,DDYGGQSTYVMDA; and humanized framework regions.
 4. A humanizedanti-idiotype antibody or fragment thereof according to claim 3, whereinthe heavy chain variable region comprises the KOLWI2VH-1 or theKOLWI2VH-2 sequence, SEQ ID NOS: 19 or 20, respectively.
 5. A humanizedanti-idiotype antibody or fragment thereof according to claim 3, whereinthe light chain variable region comprises the REIWI2VK or the REIWI2VKRSsequence, SEQ ID NOS: 21 or 23, respectively.
 6. A humanized antibody orfragment thereof according to claim 3, wherein said anti-idiotypeantibody or antibody fragment is labeled with a radiolabel, an enzyme,or a fluorescent agent.
 7. An immunogenic composition comprising theanti-idiotype antibody or antibody fragment of claim 1, conjugated to asoluble immunogenic carrier protein, for use in stimulating an immuneresponse in a patient against a cancer characterized by expression ofCEA.
 8. An immunogenic composition comprising the humanizedanti-idiotype antibody or antibody fragment of claim 3, conjugated to asoluble immunogenic carrier protein, for use in stimulating an immuneresponse in a patient against a cancer characterized by expression ofCEA.
 9. A method of clearing an antibody or antibody fragment orconjugate thereof that specifically binds CEA, wherein said antibody orantibody fragment or conjugate Thereof has been administered to saidpatient, the method comprising subsequently administering to saidpatient an anti-idiotype antibody or fragment thereof according to claim1 to clear non-targeted anti-CEA antibody or antibody fragment orconjugate thereof.
 10. A method of clearing an antibody or antibodyfragment or conjugate thereof that specifically binds CEA, wherein saidantibody or antibody fragment or conjugate thereof has been administeredto said patient, the method comprising subsequently administering tosaid patient an anti-idiotype antibody or fragment thereof according toclaim 3 to clear non-targeted anti-CEA antibody or antibody fragment orconjugate thereof.
 11. A method of stimulating an immune response in apatient against cancers expressing carcinoembryonic antigen, whichcomprises administering to said patient an effective amount of theimmunogenic composition of claim 8, conjugated to a soluble immunogeniccarrier protein, optionally in combination with a pharmaceuticallyacceptable adjuvant.
 12. A method of stimulating an immune response in apatient against cancers expressing carcinoembryonic antigen, whichcomprises administering to said patient an effective amount of theimmunogenic composition of claim 7, conjugated to a soluble immunogeniccarrier protein, optionally in combination with a pharmaceuticallyacceptable adjuvant.